Despite 31P NMR being a potentially important tool to non-invasively monitor tissue metabolism in states of health and disease, no single study has addressed the quantitative aspects of the method by comparing it to conventional chemical methods. Hearts from 200-350 g fed male Wistar rats were perfused at low and high workloads with Pi-free Krebs-Henseleit medium containing either 10 mM glucose or 10 mM glucose plus 15 mU/ml insulin. The intracellular pH by 31P NMR ranged between 6.99 and 7.02 and agreed to within 0.1 pH unit of estimates calculated using enzymatically determined total tissue HCO3-/CO2 contents. At high work, where the tissue contents of PCr and ATP were determined on the same heart as NMR areas (n=16), the proportionality factors, defined as the 31P NMR area units divided by the total enzymatic tissue content (area units/umol g-1 wet wt heart), was 112 +/- 8 for PCr; 99 +/- 4 for g-ATP; 138 +/- 9 for a-ATP and 100 +/- 4 for b-ATP. It was concluded that ATPO and PCr were equally visible by 31P NMR and that no significant breakdown of PCr from tissue acid extraction occurred. The other part of the study focused on the estimation of cytosolic Pi. The cytosolic Pi estimated from NMR in glucose-perfused hearts at low and high work was 0.92 +/- 0.11 and 0.92 +/- .08 umol/g wet wt. Using the near-equilibrium expression of KCK/KG+G and the metabolite levels in heart extracts, the calculated cytosolic Pi was 1.45 +/- 0.19 and 3.26 +/- 0.50 umol/g wet wt respectively. The cytosolic NMR Pi in the glucose plus insulin hearts was 0.54 +/- 0.07 and 0.76 +/- 0.05 umol/g wet wt at low and high work and 1.57 +/- 0.22 and 1.94 +/- 0.37 umol/g wet wt from near-equilibrium estimates. The total tissue Pi measured enzymatically ranged from 2.16 +/- 3.17 umol/g wet wt heart. The validity of using both the NMR and near-equilibrium method for estimating cytosolic Pi in the heart was discussed.